Corn Plant Event MON87460 and Compositions and Methods for Detection Thereof

ABSTRACT

The present invention provides a transgenic corn event MON87460, and cells, seeds, and plants comprising DNA diagnostic for the corn event. The invention also provides compositions comprising nucleotide sequences that are diagnostic for MON87460 in a sample, methods for detecting the presence of MON87460 event polynucleotides in a sample, and probes and primers for use in detecting nucleotide sequences that are diagnostic for the presence of MON87460 in a sample. The present invention also provides methods of breeding with MON87460 to produce water deficit tolerance corn plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/919,845 which was the National Stage of International PatentApplication No. PCT/US2009/035288, filed Feb. 26, 2009 and incorporatedby reference herein in its entirety, which claims benefit of U.S.Provisional Application Ser. No. 61/032,568 filed Feb. 29, 2008, whichis incorporated by reference herein in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“54813A_PCT.txt”, which is 19671 bytes (measured in operating systemMS-Windows), created on Feb. 4, 2009, is filed herewith by electronicsubmission and incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Disclosed herein are transgenic cells, seeds, and plants which includerecombinant DNA expressing a cold shock protein that imparts improvedstress tolerance and/or yield to plants. The disclosure also includesmethods of making, using and detecting such cells, seeds and plants. Inparticular, the present invention relates to stress tolerant corn plantsdesignated as MON87460, and methods and compositions for detecting thepresence of MON87460 DNA in a sample.

BACKGROUND OF THE INVENTION

Transgenic plants with improved agronomic traits such as yield,environmental stress tolerance, pest resistance, herbicide tolerance,improved seed compositions, and the like are desired by both farmers andseed producers. Although considerable efforts in plant breeding haveprovided significant gains in desired traits, the ability to introducespecific DNA into plant genomes provides further opportunities forgeneration of plants with improved and/or unique traits.

SUMMARY OF THE INVENTION

Compositions and methods related to transgenic water deficit stresstolerant corn plants designated MON87460, and progeny and populationsthereof are provided herein.

In one aspect, this invention provides the transgenic corn plantsdesignated MON87460 and seed of said plant as deposited with a shipmentmailed on Jan. 31, 2008 with American Type Culture Collection (ATCC) andassigned Accession No. PTA-8910. Another aspect of the inventioncomprises progeny plants, or seeds, or regenerable parts of the plantsand seeds of the plant MON87460. Progeny plants, or seeds, orregenerable parts of the plants and seeds comprising SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:7, or SEQ ID NO:24 are alsoprovided herein.

Another aspect of the invention provides polynucleotides comprising atransgene/genomic junction region from corn plant MON87460.Polynucleotides are provided that comprise at least onetransgene/genomic junction nucleic acid molecule selected from the groupconsisting of SEQ ID NO:1 through SEQ ID NO:4, SEQ ID NO:25, andcomplements thereof, wherein the junction molecule spans the transgeneinsertion site. A corn seed and plant material thereof comprising anyone of SEQ ID NO:1 through SEQ ID NO:4 or SEQ ID NO:25, is an aspect ofthis invention.

The present invention is also directed to a nucleus of a corn cell ofevent MON87460, wherein said nucleus comprises a chromosome having aheterologous polynucleotide insert that provides for improved waterdeficit tolerance, wherein said heterologous polynucleotide comprisesany one of SEQ ID NO:1 through SEQ ID NO:4. Of particular interest is achromosome wherein the heterologous polynucleotide comprises a truncatedrice actin promoter for expression of a cspB gene, and wherein saidtruncated rice actin promoter is adjacent to corn genomic sequence ofSEQ ID NO:5. In certain embodiments, a corn chromosome comprising SEQ IDNO:1 and a heterologous transgenic insert comprising a truncated riceactin promoter that is operably linked to a cspB gene is provided. A 5′terminus of the heterologous transgenic insert can overlap a 3′ terminusof SEQ ID NO:1 in certain embodiments. In certain embodiments, the cornchromosome can comprise SEQ ID NO:7 or SEQ ID NO:24. In certainembodiments, a chromosome of the invention is located within a corn cellthat also contains a second unlinked heterologous polynucleotide forexpression of a glyphosate resistant 5-enolpyruvylshikimate-3-phosphatesynthase (CP4 EPSPS) protein. Plants or seed comprising any of the cornchromosomes of the invention are also provided. Also provided are aprocessed food or feed commodity prepared from a corn seed having achromosome comprising SEQ ID NO:1 and a heterologous transgenic insertcomprising a truncated rice actin promoter that is operably linked to acspB gene, where the processed food or feed commodity comprises adetectable amount of a polynucleotide comprising a nucleotide sequenceof SEQ ID NO: 1, SEQ ID NO:2, or a complement thereof. In certainembodiments, the food or the feed commodity comprises corn meal, cornflour, corn gluten, corn oil and corn starch. In certain embodiments,the polynucleotide can comprise a nucleotide sequence of SEQ ID NO: 3,SEQ ID NO: 4, or a complement thereof. In other embodiments, thepolynucleotide can further comprise a nucleotide sequence contained inSEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:24.

According to another aspect of the invention, a pair of nucleotideprimers are used in a DNA detection method, wherein the primer pair whenused in a nucleic acid amplification method produces an amplicon thatcontains any one of SEQ ID NO:1 through SEQ ID NO:4. Detection of anyone of SEQ ID NO:1 through SEQ ID NO:4 in an amplicon produced in thismanner is diagnostic for the presence of nucleic acids from corn plantMON87460 in the sample analyzed in the detection method. Such methodscomprise: (a) contacting the sample comprising MON87460 genomic DNA witha DNA primer pair; and (b) performing a nucleic acid amplificationreaction, thereby producing an amplicon; and (c) detecting the amplicon,wherein the amplicon comprises SEQ ID NO:1 through SEQ ID NO:4.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the corn plant MON87460DNA in a sample are provided. Such methods comprising: (a) contactingthe sample comprising MON87460 DNA with a DNA probe comprising any oneof SEQ ID NO:1 through SEQ ID NO:4, or DNA molecules substantiallyhomologous to SEQ ID NO:1 through SEQ ID NO:4 that hybridize understringent hybridization conditions with genomic DNA from corn plantMON87460 and do not hybridize under stringent hybridization conditionswith non-MON87460 corn plant DNA; (b) subjecting the sample and probe tostringent hybridization conditions; and (c) detecting hybridization ofthe probe to the corn plant MON87460 DNA.

According to another aspect of the invention, methods of producing waterdeficit stress tolerant corn plants are provided and comprise the stepof crossing a first parental homozygous corn plant of event MON87460with a second parental homozygous corn plant that lacks the waterdeficit stress tolerance trait, thereby producing water deficit stresstolerant hybrid progeny plants. In certain embodiments, a method ofproducing a drought tolerant corn plant comprising crossing a droughttolerant first parent corn plant comprising a SEQ ID NO:1 and aheterologous transgenic insert comprising a truncated rice actinpromoter that is operably linked to a cspB gene, and a second parentcorn plant, thereby producing a plurality of drought tolerant progenyplants is provided. In other embodiments, the insert can comprise SEQ IDNO:7 or SEQ ID NO:24.

Another aspect of the invention is a method of determining the zygosityof the progeny of corn event MON87460 using DNA amplification reactionsand two primer sets. A first primer set is used for amplification ofMON87460 corn DNA and a second primer set is used for amplification ofnative corn sequence encompassing the transgene insertion site inMON87460 genomic DNA. Where the template for amplification is a cornplant homozygous for the MON87460 DNA an amplicon is produced only fromthe first primer set. Where the template for amplification is a cornplant heterozygous for the MON87460 DNA, amplicons are produced onlyfrom both the first primer set and the second primer set.

Also encompassed by the present invention is hybrid corn seed comprisingin its genome any one of SEQ ID NO:1 through SEQ ID NO:4 wherein atleast one parent in the cross used to create said hybrid seed isMON87460.

Other specific embodiments of the invention are disclosed in thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a plasmid map of pMON73608.

FIG. 2 illustrates the genomic organization of the transgene insert incorn plant MON87460.

FIGS. 3A,B,C provides sequence (SEQ ID NO:24) of the transgene andgenomic DNA junction region of MON87460. Corn genomic flanking DNAsequence is shown in small letters. Transgene sequence inserted frompMON73608 is shown in capital letters.

DETAILED DESCRIPTION OF THE INVENTION

A transgenic corn plant, herein referred to as “MON87460”, or “CspB-ZmEvent MON87460” is tolerant to water deficit stress as the result ofexpression of a cspB protein from E. coli in cells of said transgenicplant. Use of the water deficit stress tolerant corn will provide majorbenefits to corn growers, for example providing 5-10% higher crop yieldsin western dry-land acres where the average yearly rainfall isinsufficient to support an agriculturally effective yield from wild-typecorn plants. Additionally, MON87460 corn plants provide the benefit ofdrought insurance in central, eastern & southern corn belt by providinghigher crop yields under drought conditions as compared to wild-typecorn plants. Corn growers will also benefit from irrigation cost savingsin regions where corn is typically grown under irrigation.

As used herein, “water deficit” means a period when water available to aplant is not replenished at the rate at which it is consumed by theplant. A long period of water deficit is colloquially called droughtwhich can result in loss of a crop, even a crop enabled with thechromosomes of this invention. Lack of rain or irrigation may notproduce immediate water stress if there is an available reservoir ofground water for the growth rate of plants. Plants grown in soil withample groundwater can survive days without rain or irrigation withoutadverse affects on yield. Plants grown in dry soil are likely to sufferadverse affects with minimal periods of water deficit. Severe waterstress can cause wilt and plant death; moderate drought can causereduced yield, stunted growth or retarded development. Plants canrecover from some periods of water stress without significantlyaffecting yield. However, water stress at the time of pollination canhave an irreversible effect in lowering yield. Thus, a useful period inthe life cycle of corn for observing water stress tolerance is the latevegetative stage of growth before tasseling. The testing of water stresstolerance is often done through the comparison to control plants. Forinstance, plants of this invention can survive water deficit with ahigher yield than control plants. In the laboratory and in field trialsdrought can be simulated by giving plants of this invention and controlplants less water than an optimally-watered control plant and measuringdifferences in traits.

The corn plant MON87460 was produced by Agrobacterium mediatedtransformation of an inbred corn line with the vector pMON73608 (FIG.1). This vector contains the cspB coding region regulated by the riceactin promoter, the rice actin intron, and the tr7 3′ polyadenylationsequence, and an nptII coding region regulated by the CaMV 35S promoter,and the NOS 3′ polyadenylation sequence. Events generated from thevector pMON73608 were characterized by detailed molecular analyses.

A transgenic event in a plant occurs when recombinant DNA is insertedinto a location in a chromosome in the nucleus. It is statisticallyimprobable that any two separate transgenic events would be the same.Plants reproduced from a specific event will generally have consistencyin trait. Not all transgenic events will provide transgenic plant seed,plants, or nuclei of this invention because of a variety of factors suchas the location, copy number and integrity of the recombinant DNA in thechromosome, unintended insertion of other DNA, etc. As a result adesired transgenic event is identified by screening the transformedplant or its progeny seed for enhanced water deficit tolerance.

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet. 22:421-477, 1988). For this reason, it is often necessary toscreen a large number of plants in order to identify a plantcharacterized by optimal expression of an introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be a wide variation in levels of expression of an introducedtransgene among plants. There may also be differences in spatial ortemporal patterns of expression, for example, differences in therelative expression of a transgene in various plant tissues, that maynot correspond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. A plant that hasdesired levels or patterns of transgene expression is useful forintrogressing the transgene into other genetic backgrounds by sexualcrossing using conventional breeding methods. Progeny of such crossesmaintain the transgene expression characteristics of the originaltransformant. This strategy is used to ensure reliable gene expressionin a number of varieties that are well adapted to local growingconditions and market demands.

Events generated by transformation with pMON73608 were screened forinsert number (number of integration sites within the corn genome), copynumber (the number of copies of the T-DNA within one locus), theintegrity of the inserted cassettes and the absence of backbone sequenceusing Southern blot analyses. Probes included the intact cspB and nptIIcoding regions and their respective promoters, introns, andpolyadenylation sequences and the vector plasmid backbone. Fromapproximately 140 initial transformants, events were selected based oncopy number and backbone analysis for phenotypic analysis to identifyplants having an improved phenotype from expression of cspB. Results ofa greenhouse based test for water-deficit tolerance, identified a numberof independent transformants having water deficit tolerance. Fieldtesting of 22 selected transformants for water deficit tolerance underfield growth conditions resulted in the identification of 10 improvedevents that were further tested for water-deficit tolerance and yieldimprovement and stability. Results of these further analyses identifiedMON87460 as having superior improved phenotypes. Extensive molecularcharacterization of MON87460 demonstrated that the event contains asingle T-DNA insertion with one copy of both the cspB and nptIIcassettes. Northern blot analysis confirmed that the expected sizetranscripts for both cspB and nptII are generated in MON87460. The dataalso surprisingly demonstrate that the Agrobacterium right borderfragment is not present in MON87460 and that a truncation of the riceactin promoter regulating expression of the cspB gene has occurred suchthat only 108 bp (of 844 bp present in pMON73608) of the promoter DNA ispresent.

PCR and DNA sequence analyses were performed to determine the 5′ and 3′insert-to-plant genome junctions, confirm the organization of theelements within the insert (FIG. 2), and determine the complete DNAsequence of the insert in corn plant MON87460 (SEQ ID NO:5). Analysesconfirmed that the in planta T-DNA in MON87460 is identical tocorresponding sequence from pMON73608, Sequence analysis also identified1060 bp of 5′ and 1260 bp of 3′ flanking sequence for the MON87460insert. Comparison to the sequence of wild-type DNA of the inbred lineused for transformation showed that a 22 bp deletion of corn genomic DNAoccurred at the site of integration of the MON87460 T-DNA.

It is advantageous to be able to detect the presence oftransgene/genomic DNA of MON87460 in order to determine whether progenyof a sexual cross contain the transgene/genomic DNA of interest. Inaddition, a method for detecting MON87460 is useful when complying withregulations requiring the pre-market approval and labeling of foodsderived from the recombinant crop plants. It is possible to detect thepresence of a transgene by any well-known nucleic acid detection methodssuch as the polymerase chain reaction (PCR) or DNA hybridization usingpolynucleotide probes. These detection methods generally use DNA primerand probe molecules that are specific to the genetic elements, such aspromoters, leaders, introns, coding regions, 3′ transcriptionterminators, marker genes, etc, that are the components of thetransgenes of a DNA construct. Such methods may not be useful fordiscriminating between different transgenic events, particularly thoseproduced using the same transgene DNA construct unless the sequence ofgenomic DNA adjacent to the inserted transgene DNA is known. The presentinvention provides sequences and assays for detection of the noveltransgene/genomic DNA border junctions of MON87460.

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Generally, the nomenclature usedand the manufacture or laboratory procedures described below are wellknown and commonly employed in the art. Conventional methods are usedfor these procedures, such as those provided in the art and variousgeneral references. Unless otherwise stated, nucleic acid sequences inthe text of this specification are given, when read from left to right,in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNAor as RNA, as specified; disclosure of one necessarily defines theother, as is known to one of ordinary skill in the art. Furthermore,disclosure herein of a given nucleic acid sequence necessarily definesits complementary sequence, as is known to one of ordinary skill in theart. Where a term is provided in the singular, the inventors alsocontemplate aspects of the invention described by the plural of thatterm. The nomenclature used and the laboratory procedures describedbelow are those well known and commonly employed in the art. Where thereare discrepancies in terms and definitions used in references that areincorporated by reference, the terms used in this application shall havethe definitions given. Other technical terms used have their ordinarymeaning in the art that they are used, as exemplified by a variety oftechnical dictionaries. Definitions of common terms in molecular biologymay be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University, Press: New York, 1994.

As used herein, the term “corn” means Zea mays and includes all plantvarieties that can be bred with corn plant M0N87460.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. Transgenic progeny having the same nucleuswith either heterozygous or homozygous chromosomes for the recombinantDNA are said to represent the same transgenic event. Once a transgenefor a trait has been introduced into a plant, that gene can beintroduced into any plant sexually compatible with the first plant bycrossing, without the need for directly transforming the second plant.The heterologous DNA and flanking genomic sequence adjacent to theinserted DNA will be transferred to progeny when the event is used in abreeding program and the enhanced trait resulting from incorporation ofthe heterologous DNA into the plant genome will be maintained in progenythat receive the heterologous DNA.

The term “event” also refers to the presence of DNA from the originaltransformant, comprising the inserted DNA and flanking genomic sequenceimmediately adjacent to the inserted DNA, in a progeny that receivesinserted DNA including the transgene of interest as the result of asexual cross of one parental line that includes the inserted DNA (e.g.,the original transformant and progeny resulting from selfing) and aparental line that does not contain the inserted DNA. The term “progeny”denotes the offspring of any generation of a parent plant prepared inaccordance with the present invention. A transgenic “event” may thus beof any generation. The term “event” refers to the original transformantand progeny of the transformant that include the heterologous DNA. Theterm “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety that include theheterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking DNA from the transformed parent ispresent in the progeny of the cross at the same chromosomal location.

The present invention relates to the event MON87460 DNA, plant cells,tissues, seeds and processed products derived from MON87460. MON87460corn plants may be self-pollinated to produce inbred lines that arehomozygous for the MON87460 polynucleotides. The homozygous seed may begrown to produce homozygous progeny MON87460 event corn plants usefulfor crossing with other inbred corn plants to produce heterozygoushybrid corn seed. MON87460 hybrid corn seed can be grown to hybrid cornplants that exhibit water deficit tolerance and enhanced yield understress conditions as compared to control plants.

Products that may be derived from MON87460 include foodstuffs andcommodities produced from corn event MON87460. Such foodstuffs andcommodities are expected to contain polynucleotides that, if detected insufficient levels are diagnostic for the presence of corn event MON87460materials within such commodities and foodstuffs. Examples of suchfoodstuffs and commodities include but are not limited to corn oil, cornmeal, corn flour, corn gluten, corn cakes, corn starch, and any otherfoodstuff intended for consumption as a food source by an animal orotherwise, intended as a bulking agent, or intended as a component in amakeup composition for cosmetic use, etc.

It is also to be understood that two different transgenic plants can bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes.Alternatively, inbred lines containing the individual exogenous genesmay be crossed to produce hybrid seed that is heterozygous for eachgene, and useful for production of hybrid corn plants that exhibitmultiple beneficial phenotypes as the result of expression of each ofthe exogenous genes. Descriptions of breeding methods that are commonlyused for different traits and crops can be found in various references,e.g., Allard, “Principles of Plant Breeding,” John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98, 1960; Simmonds, “Principles of CropImprovement,” Longman, Inc., NY, 369-399, 1979; Sneep and Hendriksen,“Plant Breeding Perspectives,” Wageningen (ed), Center for AgriculturalPublishing and Documentation, 1979. Of particular interest in thepresent invention is the development of MON87460 event corn plants thatexpress cspB protein and a glyphosate resistant5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein (U.S.Pat. No. 5,633,435) from Agrobacterium sp. strain CP4 that confers planttolerance to glyphosate. a. “Glyphosate” refers toN-phosphonomethylglycine and its salts. N-phosphonomethylglycine is awell-known herbicide that has activity on a broad spectrum of plantspecies. Glyphosate is the active ingredient of Roundup® (Monsanto Co.),a safe herbicide having a desirably short half-life in the environment.Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Treatments with “glyphosate herbicide” refer to treatments withthe Roundup®, Roundup Ultra®, Roundup Pro® herbicide or any otherherbicide formulation containing glyphosate. Examples of commercialformulations of glyphosate include, without restriction, those sold byMonsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX,ROUNDUP®WEATHERMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE,ROUNDUP® BIOFORCE, RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, allof which contain glyphosate as its isopropylammonium salt; those sold byMonsanto Company as ROUNDUP® DRY and RIVAL® herbicides, which containglyphosate as its ammonium salt; that sold by Monsanto Company asROUNDUP® GEOFORCE, which contains glyphosate as its sodium salt; andthat sold by Syngenta Crop Protection as TOUCHDOWN® herbicide, whichcontains glyphosate as its trimethylsulfonium salt. When applied to aplant surface, glyphosate moves systemically through the plant.Glyphosate is phytotoxic due to its inhibition of the shikimic acidpathway, which provides a precursor for the synthesis of aromatic aminoacids. Glyphosate inhibits the enzyme 5-enolpyruvyl-3-phosphoshikimatesynthase (EPSPS) found in plants. Glyphosate tolerance can be achievedby the expression of bacterial EPSPS variants and plant EPSPS variantsthat have lower affinity for glyphosate and therefore retain theircatalytic activity in the presence of glyphosate (U.S. Pat. Nos.5,633,435, 5,094,945, 4,535,060, and 6,040,497).

As used herein when referring to an “isolated DNA molecule”, it isintended that the DNA molecule be one that is present, alone or incombination with other compositions, but not within its naturalenvironment. For example, a coding sequence, intron sequence,untranslated leader sequence, promoter sequence, transcriptionaltermination sequence, and the like, that are naturally found within theDNA of a corn genome are not considered to be isolated from the corngenome so long as they are within the corn genome. However, each ofthese components, and subparts of these components, would be “isolated”within the scope of this disclosure so long as the structures andcomponents are not within the corn genome.

For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of the corn plant event MON87460 would be considered to be anisolated nucleotide sequence whether it is present within the plasmidused to transform corn cells from which the MON87460 event arose, withinthe genome of the event MON87460, present in detectable amounts intissues, progeny, biological samples or commodity products derived fromthe event MON87460. The nucleotide sequence or any fragment derivedtherefrom would be considered to be isolated or isolatable if the DNAmolecule can be extracted from cells, or tissues, or homogenate from aplant or seed or plant organ; or can be produced as an amplicon fromextracted DNA or RNA from cells, or tissues, or homogenate from a plantor seed or plant organ, any of which is derived from such materialsderived from the event MON87460. For that matter, the junction sequencesas set forth at SEQ ID NO:1 and SEQ ID NO:2, and nucleotide sequencesderived from event MON87460 that also contain these junction sequencesare considered to be isolated or isolatable, whether these sequences arepresent within the genome of the cells of event MON87460 or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the event MON87460.

As used herein, a transgene/genomic junction is the point at whichheterologous DNA from a transformation vector that is inserted into thegenome is linked to the corn plant genomic DNA. A junctionpolynucleotide spans the transgene/genomic junction, and is novel in anyparticular transgenic plant event. Thus, detection of a junctionpolynucleotide in a biological sample is diagnostic for the presence ofa specific plant event. In the present invention, the presence of SEQ IDNO:1 through SEQ ID NO:4 junction polynucleotides in a sample isdiagnostic for the presence of MON87460 DNA in a sample.

A “probe” is a polynucleotide to which is attached a conventionaldetectable label or reporter molecule, e.g., a radioactive isotope,ligand, chemiluminescent agent, or enzyme. Probes are complementary to astrand of a target nucleic acid, in the case of the present invention,to a strand of genomic DNA from MON87460, whether from a MON87460 plantor from a sample that includes MON87460 DNA. Probes according to thepresent invention include not only deoxyribonucleic or ribonucleicacids, but also polyamides and other probe materials that bindspecifically to a target DNA sequence and can be used to detect thepresence of that target DNA sequence.

DNA primers are isolated polynucleotides that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. A DNAprimer pair or a DNA primer set of the present invention refer to twoDNA primers useful for amplification of a target nucleic acid sequence,e.g., by the polymerase chain reaction (PCR) or other conventionalpolynucleotide amplification methods.

DNA probes and DNA primers are generally 11 polynucleotides or more inlength, often 18 polynucleotides or more, 24 polynucleotides or more, or30 polynucleotides or more. Such probes and primers are selected to beof sufficient length to hybridize specifically to a target sequenceunder high stringency hybridization conditions. Preferably, probes andprimers according to the present invention have complete sequencesimilarity with the target sequence, although probes differing from thetarget sequence that retain the ability to hybridize to target sequencesmay be designed by conventional methods.

Methods for preparing and using polynucleotide probes and primers aredescribed, for example, in Molecular Cloning: A Laboratory Manual, 2nded., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”);Current Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing and Wiley-Interscience, New York, 1992 (with periodicupdates) (hereinafter, “Ausubel et al., 1992”); and Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, 1990. PCR DNA primer pairs can be derived from a known sequence,for example, by using computer programs intended for that purpose suchas Primer (Version 0.5,© 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.).

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA molecule. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic plant in a sample.Polynucleic acid molecules, also referred to as nucleic acid segments,or fragments thereof are capable of specifically hybridizing to othernucleic acid molecules under certain circumstances. As used herein, twopolynucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibit complete complementarity. As used herein, molecules aresaid to exhibit “complete complementarity” when every nucleotide of oneof the molecules is complementary to a nucleotide of the other. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional“high-stringency” conditions. Conventional stringency conditions aredescribed by Sambrook et al., 1989, and by Haymes et al., In: NucleicAcid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985), Departures from complete complementarity are thereforepermissible, as long as such departures do not completely preclude thecapacity of the molecules to form a double-stranded structure. In orderfor a nucleic acid molecule to serve as a primer or probe it need onlybe sufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed.

In a preferred embodiment, a polynucleotide of the present inventionwill specifically hybridize to one or more of the nucleic acid moleculesset forth in SEQ ID NO: 1-7 or complements or fragments thereof undermoderately stringent conditions, for example at about 2.0×SSC and about65° C. In a particularly preferred embodiment, a nucleic acid of thepresent invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NOs: 1-7 or complements orfragments thereof under high stringency conditions.

As used herein, “amplified DNA” or “amplicon” refers to thepolynucleotides that are synthesized using amplification techniques,such as PCR. The term “amplicon” as used herein specifically excludesprimer dimers that may be formed in a DNA amplification reaction.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction. The term “specific for (a targetsequence)” indicates that a probe or primer hybridizes under stringenthybridization conditions only to the target sequence in a samplecomprising the target sequence.

A member of a primer pair derived from the plant genomic sequenceadjacent to the transgene insert DNA is located a distance from theinserted DNA sequence, this distance can range from one nucleotide basepair up to about twenty thousand nucleotide base pairs. Similarly, amember of a primer pair derived from the transgene insert DNA is locateda distance from the plant genomic sequence junction, this distance canrange from one nucleotide base pair up to about the full length of thetransgene insert. The amplicon may range in length from the combinedlength of the primer pair plus one nucleotide base pair, but ispreferably about fifty nucleotide base pairs or longer, for example, upto 500 or even 1000 nucleotides in length. Smaller sized amplicons ingeneral are more reliably produced in PCR reactions, allow for shortercycle times and can be easily separated and visualized on agarose gelsor adapted for use in TaqMan assays, such as end-point and RealTimeTaqman. Alternatively, a primer pair can be derived from genomicsequence on both sides of the inserted heterologous DNA so as to producean amplicon that includes the entire insert polynucleotide sequence(e.g., a forward primer isolated from SEQ ID NO:5 and a reverse primerisolated from SEQ ID NO:6 that amplifies a DNA molecule comprising thepMON73608 DNA fragment that was inserted into the MON87460 genome, theinsert comprising about 3309 nucleotides (SEQ ID NO:7), shown as capitalletters in FIG. 3.

To determine whether a corn plant resulting from a sexual cross containstransgenic plant genomic DNA from the corn plant MON87460 plant of thepresent invention, DNA that is extracted from a corn plant tissue sampleis subjected to a polynucleotide amplification method using a primerpair that includes a first primer derived from DNA sequence in thegenome of the MON87460 plant adjacent to the insertion site of theinserted heterologous DNA (transgene DNA), and a second primer derivedfrom the inserted heterologous DNA to produce an amplicon that isdiagnostic for the presence of the MON87460 plant DNA. The diagnosticamplicon is of a specific length depending on the location of theprimers, and comprises a specific junction polynucleotide sequence thatis diagnostic for the specific plant event genomic DNA. The presence ofthe junction polynucleotide sequence in an amplicon can be determined,for example, by sequencing the amplicon DNA or by hybridization with aspecific probe. In the present invention, the DNA sequence of theamplicon diagnostic for the presence of the MON87460 comprises SEQ IDNO:1 or SEQ ID NO:2. More specifically, in an embodiment of the presentinvention, an amplicon diagnostic for the presence of the MON87460 is 68nt in length and comprises SEQ ID NO:2, and may be detected byhybridization with a labeled probe comprising any one of SEQ ID NO:2,SEQ ID NO:10 or SEQ ID NO:16.

Polynucleotide amplification can be accomplished by any of the variousamplification methods known in the art, including the polymerase chainreaction (PCR). Amplification methods are known in the art and aredescribed, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and inPCR Protocols: A Guide to Methods and Applications, ed. Innis et al.,Academic Press, San Diego, 1990. PCR amplification methods have beendeveloped to amplify up to 22 kb (kilobase) of genomic DNA and up to 42kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA91:5695-5699, 1994). These methods as well as other methods known in theart of DNA amplification may be used in the practice of the presentinvention. The sequence of the heterologous DNA insert or flankinggenomic DNA sequence from MON87460 can be verified (and corrected ifnecessary) by amplifying such DNA molecules from the MON87460 seed orplants grown from the seed deposited with the ATCC having accession no.PTA-8910, using primers derived from the sequences provided herein,followed by standard DNA sequencing of the PCR amplicon or cloned DNAfragments thereof.

DNA detection kits that are based on DNA amplification methods containDNA primer molecules that hybridize specifically to a target DNA andamplify a diagnostic amplicon under the appropriate reaction conditions.Any length amplicon produced from MON87460 DNA wherein the ampliconcomprises SEQ ID NO:1 or SEQ ID NO:2 is an aspect of the invention. Theskilled artisan will recognize that the first and second DNA primermolecules are not required to consist only of DNA but may also becomprised exclusively of RNA, a mixture of DNA and RNA, or a combinationof DNA, RNA, or other nucleotides or analogues thereof that do not actas templates for one or more polymerases. In addition, the skilledartisan will recognize that a probe or a primer as set forth hereinshall be at least from about 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20consecutive nucleotides in length and selected from the group ofnucleotides as set forth in SEQ ID NO:1 and SEQ ID NO:3 (arbitrarilydesignated 5′ junction), SEQ ID NO:2 and SEQ ID NO:4 (arbitrarilydesignated 3′ junction), SEQ ID NO:5 (arbitrarily designated 5′ flankingsequence), SEQ ID NO:6 (arbitrarily designated 3′ flanking sequence),and SEQ ID NO:7 (inserted transgene sequence). Probes and primers atleast from about 21 to about 50 or more consecutive nucleotides inlength are possible when selected from the group of nucleotides as setforth in SEQ ID NO:5 through SEQ ID NO:7.

The kit may provide an agarose gel based detection method or any numberof methods of detecting the diagnostic amplicon that are known in theart. A kit that contains DNA primers that are homologous orcomplementary to any portion of the corn genomic region of SEQ ID NO:5or SEQ ID NO:6 and to any portion of the transgene insert region of SEQID NO:7 is an object of the invention. Specifically identified as auseful primer pair in a DNA amplification method is SEQ ID NO:8 and SEQID NO:9 that amplify a diagnostic amplicon homologous to a portion ofthe 5′ transgene/genome region of MON87460, wherein the ampliconcomprises SEQ ID NO:2. Other DNA molecules useful as DNA primers can beselected from the disclosed transgene/genomic DNA sequence of MON87460by those skilled in the art of DNA amplification.

The diagnostic amplicon produced by these methods may be detected by aplurality of techniques. One such method is Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNAoligonucleotide is designed that overlaps both the adjacent flankinggenomic DNA sequence and the inserted DNA sequence. The oligonucleotideis immobilized in wells of a microtiter plate. Following PCR of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded PCR productcan be hybridized to the immobilized oligonucleotide and serve as atemplate for a single base extension reaction using a DNA polymerase andlabelled dideoxynucleotide triphosphates (ddNTPs) specific for theexpected next base. Readout may be fluorescent or ELISA-based. A signalindicates presence of the transgene/genomic sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. DNTPs are added individually and theincorporation results in a light signal that is measured. A light signalindicates the presence of the transgene/genomic sequence due tosuccessful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedthat overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene/genomic sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully described in the instructions provided by the manufacturer.Briefly, a FRET (fluorescence resonance energy transfer) oligonucleotideprobe is designed that overlaps the genomic flanking and insert DNAjunction. The FRET probe and PCR primers (one primer in the insert DNAsequence and one in the flanking genomic sequence) are cycled in thepresence of a thermostable polymerase and dNTPs. Hybridization of theFRET probe results in cleavage and release of the fluorescent moiety,such as 6FAM™ and VIC™, away from the quenching dye, such as TAMRA(tetramethyl-6-carboxyrhodamine) for conventional probes, ornon-fluorescent minor groove binding compounds for MGB probes. Witheither TAMRA or MGB probes, the polymerase cleaves bound probe duringPCR, separating the fluorophore and quencher to the extent that FRETcannot occur, and a fluorescent signal indicates the presence of thetransgene/genomic sequence.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal indicates thepresence of the flanking/transgene insert sequence due to successfulamplification and hybridization.

Other described methods, such as microfluidics (U.S. Patent PublicationNo. 2006068398, U.S. Pat. No. 6,544,734) may be used to separate andamplify DNA samples. Optical dyes are used to detect and quantitatespecific DNA molecules (WO105017181). Nanotube devices have beendescribed (WO/06024023) that comprise an electronic sensor for thedetection of DNA molecules or nanobeads that bind specific DNAmolecules.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for identification of corn plant MON87460 DNA in a sample andcan be applied to methods for breeding corn plants containing MON87460DNA. A kit contains DNA molecules that are useful as primers or probesand that are homologous or complementary to at least a portion of SEQ IDNO:1-7. The DNA molecules can be used in DNA amplification methods (PCR)or as probes in nucleic acid hybridization methods such as Southernanalysis and northern analysis.

In another aspect of the present invention, a preferred polynucleotideof the present invention that is diagnostic for the presence of MON87460DNA has the sequence set forth in SEQ ID NO:1 through SEQ ID NO:4, orSEQ ID NO:25. SEQ ID NO:1 through SEQ ID NO:4 and largergenomic/transgene junction polynucleotides, such as those in SEQ ID NO:5-7 may also be used as markers in plant breeding methods to identifythe progeny of genetic crosses similar to the methods described forsimple sequence repeat DNA marker analysis, in “DNA markers: Protocols,applications, and overviews: (1997) 173-185, Cregan, et al., eds.,Wiley-Liss NY. The hybridization of the probe to the target DNA moleculecan be detected by any number of methods known to those skilled in theart, these can include, but are not limited to, fluorescent tags,radioactive tags, antibody based tags, and chemiluminescent tags. Inanother aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention shares between 80% and 100% or 90% and100% sequence identity with a nucleic acid sequence set forth in SEQ IDNO:1 through SEQ ID NO:7 or complements thereof or fragments of either.In a further aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares between 95% and 100%sequence identity with a sequence set forth in SEQ ID NO:1 through SEQID NO:7 or complements thereof or fragments of either.

Water deficit tolerant corn plants that lack a selectable marker gene orlack an intact selectable marker gene also provided herein. Such plantscan be obtained by methods that comprise exposing a corn chromosomecomprising a heterologous transgene insert that confers water deficittolerance and a selectable marker gene to one or morerecombination-inducing agents and selecting a corn plant comprising aheterologous transgene insert that confers water deficit tolerance wherethe selectable marker gene has been either completely or partiallyeliminated or where the selectable marker gene has been disrupted.Heterologous transgene inserts that confer water deficit tolerance andcontain a selectable marker include, but are not limited to, insertscomprising SEQ ID NO:7 or inserts comprising SEQ ID NO:1, a truncatedrice actin promoter that is operably linked to a cspB gene, a selectablemarker gene, and SEQ ID NO:2. Corn chromosomes that comprising aheterologous transgene insert that confers water deficit tolerance and aselectable marker gene also include, but are not limited to, a cornchromosome that comprises SEQ ID NO:24, a corn chromosome of a cornplant having been deposited under ATCC Accession No. PTA-8910 (ATCC,10801 University Blvd., Manassas, Va., USA), and progeny thereof.Heterologous transgene inserts that confer water deficit toleranceinclude, but are not limited to, inserts comprising SEQ ID NO:1 and atruncated rice actin promoter that is operably linked to a cspB gene aswell as inserts comprising SEQ ID NO:1 and a truncated rice actinpromoter that is operably linked to a cspB gene where a 5′ terminus ofthe insert overlaps a 3′ terminus of SEQ ID NO:1.

The phrase “operably linked” as used herein refers to the joining ofnucleic acid sequences such that one sequence can provide a requiredfunction to a linked sequence. In the context of a promoter, “operablylinked” means that the promoter is connected to a sequence of interestsuch that the transcription of that sequence of interest is controlledand regulated by that promoter. When the sequence of interest encodes aprotein and when expression of that protein is desired, “operablylinked” means that the promoter is linked to the sequence in such a waythat the resulting transcript will be efficiently translated. If thelinkage of the promoter to the coding sequence is a transcriptionalfusion and expression of the encoded protein is desired, the linkage ismade so that the first translational initiation codon in the resultingtranscript is the initiation codon of the coding sequence.Alternatively, if the linkage of the promoter to the coding sequence isa translational fusion and expression of the encoded protein is desired,the linkage is made so that the first translational initiation codoncontained in the 5′ untranslated sequence associated with the promoterand is linked such that the resulting translation product is in framewith the translational open reading frame that encodes the proteindesired. Nucleic acid sequences that can be operably linked include, butare not limited to, sequences that provide gene expression functions(i.e., gene expression elements such as promoters, 5′ untranslatedregions, introns, protein coding regions, 3′ untranslated regions,polyadenylation sites, and/or transcriptional terminators), sequencesthat provide DNA transfer and/or integration functions (i.e., T-DNAborder sequences, site specific recombinase recognition sites, integraserecognition sites), sequences that provide for selective functions(i.e., antibiotic resistance markers, biosynthetic genes), sequencesthat provide scoreable marker functions (i.e., reporter genes),sequences that facilitate in vitro or in vivo manipulations of thesequences (i.e., polylinker sequences, target sequences for sitespecific recombinases) and sequences that provide replication functions(i.e., bacterial origins of replication, autonomous replicationsequences, centromeric sequences).

Recombination inducing agents can comprise ionizing radiation and/or anycompound, protein, and/or a nucleic acid that provides for eliminationor modification of a polynucleotide sequence. Recombination inducingagents thus include, but are not limited to, agents that provide forhomologous recombination, non-homologous recombination, site-specificrecombination, and/or genomic modifications. Genomic modificationsprovided by recombination inducing agents thus include, but are notlimited to, insertions, deletions, inversions, and/or nucleotidesubstitutions. Use of recombination inducing agents to induce geneticmodifications in plants has been disclosed (Lloyd et al., Proc Natl AcadSci USA., 102(6):2232, 2005). Recombination agents can be native orengineered. Site specific recombinases include, but are not limited to,a Cre-recombinase, a FLP recombinase, a Flippase and the like.Recombination-inducing agents also include, but are not limited tonucleases. Nucleases that can be used include, but are not limited to,meganucleases and zinc-finger nucleases. Other recombination-inducingagents include, but are not limited to, homologous replacement sequencesand non-homologous replacement sequences. In certain embodiments,recombination-inducing reagents can comprise a nuclease and a homologousor non-homologous replacement sequence. In certain embodiments, acre-recombinase capable of excising the selectable marker locatedbetween the lox sites of SEQ ID NO:24 can be used. Cre-mediatedelimination of sequences flanked by lox sites in plants has beendisclosed (U.S. Pat. No. 5,658,772).

Elimination or disruption of a selectable marker gene, a portionthereof, or other sequence can be effected by inducing a double strandedbreak in the target sequence, providing a homologous replacementsequence that lacks the selectable marker gene or a portion thereof, andrecovering plants where the replacement sequence has integrated in placeof the originally resident sequences. A homologous replacement sequencecan comprise homologous sequences at both ends of the double strandedbreak that are provide for homologous recombination and substitution ofthe resident sequence in the chromosome with the replacement sequence.Targeted double-strand break-induced homologous recombination in cropplants such as tobacco and maize has been disclosed (Wright et al.,Plant J. 44, 693, 2005; D'Halluin, et al., Plant Biotech. J. 6:93,2008). It is also possible to insert a homologous replacement sequenceinto a targeted nuclease cleavage site by non-homologous end joining ora combination of non-homologous end joining and homologous recombination(reviewed in Puchta, J. Exp. Bot. 56, 1, 2005). Targeted insertion ofhomologous replacement sequences into specific plant genomic sites bynon-homologous end joining or a combination of non-homologous endjoining and homologous recombination has also been disclosed (Wright etal., Plant J. 44, 693, 2005). In certain embodiments, a meganucleasethat catalyzes at least one site specific double stranded break in theselectable marker gene can be used. Meganucleases have been shown to beamenable to genetic modification such that they can be evolved orengineered (WO/06097853A1, WO/06097784A1, WO/04067736A2) or rationallydesigned (U.S. 20070117128A1) to cut within a recognition sequence thatexactly matches or is closely related to specific target sequence. Inthese cases, given a reasonably sized target such as a selectable markergene sequence, one can select or design a nuclease that will cut withinthe target selectable marker gene sequence. Alternatively, a zinc fingernuclease that that catalyzes at least one site specific double strandedbreak in the selectable marker gene can be used. Such zinc-fingernucleases, the ability to engineer specific zinc-finger nucleases, andtheir use in providing for homologous recombination in plants have alsobeen disclosed (WO 03/080809, WO 05/014791, WO 07014275, WO 08/021,207).

Elimination or disruption of a selectable marker gene, a portionthereof, or other sequence can also be effected by inducing a doublestranded break in the target sequence, providing a non-homologousreplacement sequence that lacks the selectable marker gene or a portionthereof, and recovering plants where the non-homologous replacementsequence has integrated in the target sequence. In certain embodiments,a non-homologous replacement sequence can comprise single strandedsequences at both ends that are complementary to single strandedsequences at both ends of the double stranded break to provide fornon-homologous end joining of the replacement sequence and doublestranded break.

Methods for de novo generation of a corn plant that is substantiallyequivalent to a corn plant of event MON87460 and resultant plants arealso provided herein. Such methods can comprise use ofrecombination-inducing agents. Corn plants that are substantiallyequivalent to a corn plant of event MON87460 include, but are notlimited to, corn plants comprising a chromosome having a heterologoustransgenic insert comprising a promoter that is operably linked to acspB gene, where the transgenic insert is present at the same orsubstantially the same chromosomal location or chromosomal integrationsite as in MON87460. Promoters that can be operably linked to a cspBgene, include, but are not limited to, rice actin promoters, includingtruncated rice actin promoters, a maize RS81 promoter, a maize RS324promoter, a maize A3 promoter, viral promoters, and the like. In certainnon-limiting embodiments, one can select, evolve or design a nuclease tocut within a target recognition sequence that exactly matches or isclosely related to a target sequence in SEQ ID NO:5, in SEQ ID NO:6, ina corn chromosomal sequence that spans SEQ ID NO:5 and SEQ ID NO:6 in anon-transgenic corn plant, or in SEQ ID NO:23. In these or othernon-limiting embodiments, a genetically-modified corn plant whichcontains a promoter operably linked to a cspB gene can be produced by:i) introducing into a corn plant cell a homologous replacement sequencecomprising a promoter that is operably linked to a cspB gene andflanking sequences that are substantially identical to a target sequenceand a nuclease that cleaves the target sequence; and ii) selecting for acorn cell or corn plant where the homologous replacement sequence hasintegrated into the target sequence. Given that corn chromosomal targetsequences disclosed herein have been found to be favorable sites fortransgene insertion, methods for obtaining plants with insertions of oneor more transgenes that confer traits other than water deficit toleranceor transgenes that comprise genes other than cspB that confer waterdeficit tolerance into target sites disclosed herein are also provided.

The availability of recombination-inducing agents and various homologousreplacement sequences also provides for water deficit tolerant cornplants that comprise one or more additional gene(s) integrated into thesame chromosomal location as the heterologous transgene insert thatconfers water deficit tolerance. Integration of the additional genes atthe same location as the gene that confers water deficit tolerance isadvantageous in that any traits carried by the additional genes will begenetically linked to the water deficit tolerance trait, thusfacilitating breeding. In certain embodiments, an additional gene orgenes can be a gene or genes that work in concert with the residentheterologous transgene insert that confers water deficit tolerance toprovide additional water deficit tolerance. In certain embodiments, anadditional gene or genes can be a gene or genes that provide a distinctand useful trait other than water deficit tolerance. Thus, one or moregenes that confer one or more traits include, but are not limited to,genes that confer herbicide resistance, pest resistance, improved yieldunder water sufficient conditions, improved seed oil, improved seedstarch, improved seed protein, and/or improved nitrogen utilization.Such plants can be obtained by methods that comprise exposing a cornchromosome comprising a heterologous transgene insert that confers waterdeficit tolerance to a homologous replacement sequence comprising one ormore additional genes and selecting a corn plant comprising aheterologous transgene insert that confers water deficit tolerance andone or more additional genes. In certain embodiments, the insertion ofthe homologous replacement sequence can be facilitated by use of anadditional recombination-inducing agent. Additionalrecombination-inducing agents used include thus include, but are notlimited to, a meganuclease, a zinc-finger nuclease, or other agent thatinduces a double-stranded break at a desired site of double-strandbreak-induced homologous recombination. Heterologous transgene insertsthat confer water deficit tolerance include, but are not limited to,inserts comprising SEQ ID NO:1 and a truncated rice actin promoter thatis operably linked to a cspB gene as well as inserts comprising SEQ IDNO:1 and a truncated rice actin promoter that is operably linked to acspB gene where a 5′ terminus of the insert overlaps a 3′ terminus ofSEQ ID NO:1. In certain embodiments, the homologous replacement sequencecomprises a sequence that provides for replacement of a selectablemarker gene that is in a resident heterologous transgene insert with oneor more additional genes. Heterologous transgene inserts that conferwater deficit tolerance and contain a selectable marker include, but arenot limited to, inserts comprising SEQ ID NO:7 or inserts comprising SEQID NO:1 and a truncated rice actin promoter that is operably linked to acspB gene. Corn chromosomes that comprise a heterologous transgeneinsert that confers water deficit tolerance and a selectable marker genealso include, but are not limited to, a corn chromosome that comprisesSEQ ID NO:24, a corn chromosome of corn plant having been depositedunder ATCC Accession No. PTA-8910, and progeny thereof. In certainembodiments, an additional gene or gene can also be inserted into SEQ IDNO:5 and/or SEQ ID NO:6.

It is also anticipated that any of the aforementioned additional gene orgenes can be integrated into a chromosome comprising SEQ ID NO:1 and atruncated rice actin promoter that is operably linked to a cspB gene andone or more lox sites by site specific recombination. Site specificrecombination systems used for this purpose include, but are notlimited, to FLP recombinase/FRT, cre recombinase/lox, and combinationsthereof. The use of site-specific recombination systems in plants andother eukaryotic organisms has been disclosed (U.S. Pat. No. 5,801,030,U.S. Pat. No. 5,658,772, and U.S. Pat. No. 6,262,341): The presence oflox site specific recombination sites in corn chromosomes comprising SEQID NO:7 or SEQ ID NO:24 and a corn chromosome of a corn plant havingbeen deposited under ATCC Accession No. PTA-8910, and progeny thereof,thus provides for site specific integration of additional genes intothese corn chromosomes. In certain embodiments, the selectable markersequence which is flanked by the lox sites in the corn chromosomes isfirst excised by cre-recombinase, leaving a single lox site in thechromosome. Additional genes can then be introduced on a circular DNAmolecule comprising the additional genes and an operably linked lox siteand integrated into the corn chromosome at the single lox site that wasleft in the chromosome. Exemplary schemes for creating circular DNAmolecules and site-specific integration of genes into chromosomes havebeen disclosed (Vergunst et al., Nucleic Acid Res. 26(11), 279, 1998).Introduction of site-specific recombination sites other than lox at thechromosomal location of the SEQ ID NO:24 insertion and insertion ofadditional genes at those recombination sites is also provided herein.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found to functionwell in the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Production of Transgenic Corn Plants

Transgenic corn plants were produced by Agrobacterium. mediatedtransformation of LH59 corn with the vector pMON73608 (FIG. 1). Thisvector contains the cspB coding region regulated by the rice actinpromoter, the rice actin intron, and the tr7 3′ polyadenylationsequence, and the nptII coding region regulated by the 35S CaMVpromoter, and the NOS 3′ polyadenylation sequence.

TABLE 1 Summary of Genetic Elements in pMON73608 Position in GeneticElement FIG. 1 Function (Reference) CR-Ec.rop-1:1:3  53-244 Codingsequence for repressor of primer protein OR-Ec.ori-ColE1-1:1:1 672-1260Origin of replication from pBR322 for maintenance of plasmid in E. coliP-Ec.aadA-SPC/STR-1:1:1 1793-2681 Bacterial promoter and coding sequencefor an CR-Ec.aadA-SPC/STR-1:1:3 aminoglycoside-modifying enzyme, 3′(9)-T-Ec.aadA-SPC/STR-1:1:1 Onucleotidyltransferase from the transposon Tn7(GenBank accession X03043) B-AGRtu.right border- 2816-3172 Right bordersequence essential for transfer of T-DNA 1:1:12 derived fromAgrobacterium P-Os.Act1-1:1:8 3205-4048 Promoter, leader and intron fromthe rice actin gene L-Os.Act1-1:1:5 4049-4128 I-Os.Act1-1:1:3 4129-4605CR-Bs.cspB-1:4:1 4608-4811 Coding region for the CSPB protein fromBacillus subtilis which a change in the second amino acid position fromleucine to valine (WO05033318) T-AGRtu.tr7-1:1:5 4842-5349 3′nontranslated region of the transcript 7 coding sequence fromAgrobacterium that directs polyadenylation RS-P1.1ox1-1:1:1 5424-5457Recombination site recognized by Cre recombinase P-CaMV.35S-1:1:65484-5776 Cauliflower mosaic virus (CaMV) promoter CR-Ec.nptII-Tn5-1:1:35841-6635 Coding region isolated from Tn5 which codes for neomycinphosphotransferase type II. Expression of this gene in plant cellsconfers resistance to kanamycin and serves as a selectable marker fortransformation T-AGRtu.nos-1:1:13 6667-6919 3′ nontranslated region ofthe nopaline synthase (NOS) coding sequence from Agrobacteriumtumifaciens that directs polyadenylation RS-P1.1ox1-1:1:1 6945-6978Recombination site recognized by Cre recombinase B-AGRtu.leftborder-1:1:5 6999-7440 Left border sequence essential for transfer ofT-DNA derived from Agrobacterium OR-Ec.oriV-RK2-1:1:6 7527-7923 Originof replication for Agrobacterium derived from the broad host rangeplasmid RK2

LH59 callus was initiated from immature embryos. Immature embryos, 1.5mm to 2.0 mm, were excised from developing maize plants and culturedwith the embryonic axis side down on callus initiation medium for 8-21days.

Agrobacterium was prepared via standard methods and 50 to 100 pieces ofcallus were transferred to a Petri dish containing about 15 ml ofAgrobacterium suspension at 0.1 to 1.0×109 cfu/ml. Callus pieces, 2 mmto 8 mm in diameter, were incubated for about 30 minutes at roomtemperature with the Agrobacterium suspension, followed by removal ofthe liquid by aspiration. About 50 uL of sterile distilled water wasadded to filter paper in a 60×20 mm Petri dish. Fifteen to 20 pieces ofinoculated callus were transferred to each filter paper and the platesealed. The callus and Agrobacterium were co-cultured for about 3 daysat 23° C. in the dark.

Calli were transferred from filter paper to medium callus initiationmedium containing carbenicillin and cultured in the dark at 27° C. to28° C. for 2-5 days. Selection was initiated by transferring callus tocallus initiation medium containing silver nitrate, carbenicillin andmg/L paromomycin. After 2 weeks culture in the dark at 27° C. to 28° C.,callus was transferred to medium containing higher levels ofparomomycin. Callus was subcultured after two weeks to fresh medium andfurther cultured for two weeks in the dark at 27° C. to 28° C. Calluswas then transferred to again to medium with higher levels ofparomomycin. After 2-3 weeks culture in the dark at 27° C. to 28° C.,paromomycin resistant callus was identified.

Plants were regenerated (RO plants) from transformed callus, transferredto soil and grown in the greenhouse. RO plants were screened by PCR forpresence of the cspB and nptII coding regions, and Southern analysis wasconducted to determine insert copy. Taqman analysis was used todetermine presence or absence of vector backbone sequences. Transgenicevents that were positive for the presence of both cspB and nptII genes,negative for the presence of vector backbone sequences and had one ortwo inserts (integration sites within the corn genome) were selected forphysiological analysis for drought tolerance. The positive events weregrown in the greenhouse to maturity and selfed. Homozygous,heterozygous, and non-transgenic seed from multiple transgenic eventsobtained by genomic insertions of the T-DNA of pMON73608 were collectedfrom the selfed positive plants.

Example 2 Greenhouse Screening for Water Deficit Stress Tolerance

Transgenic heterozygous corn plants were grown from the heterozygousseed from transgenic events transformed with pMON73608 (Example 1) andscreened for water deficit stress tolerance as compared to controlplants by a high-throughput method of greenhouse screening in whichwater is withheld to create a “drought treatment”. Water use efficiencyis measured by plant growth rate, e.g., at least a 10% improvement, inheight and biomass during a drought treatment, as compared to controlplants. The hydration status of the shoot tissues following the droughtis also measured. Shoot Initial Height (SIH) is plant height after 3weeks of growth under optimum conditions. Shoot Wilt Height (SWH) isplant height at the end of a 6 day drought. Time course experiments haveshown that at about 3 days of drought treatment, wild type corn plantsbasically stop growing and begin to wilt. Thus, a transgenic corn plantwith improved water use efficiency will continue to grow (althoughpossibly to a lesser extent than with water) and thereby besignificantly taller at the end of a drought experiment. Shoot Wilt Mass(SWM) is the amount of wet and dry matter in the shoot (plant separatedfrom root ball at the soil line) at the end of the drought; SDM ismeasured after 2 to 3 weeks in a drying chamber. Shoot Turgid mass (STM)is the SWM plus the mass of the water that is transported into planttissues in 3 days of soaking in 40 degree Celsius water in the dark.Experiments have shown that most of the water is pulled up in 24 hoursbut it takes 2 more days before additional increase becomesinsignificant. STM−SWM is indicative of water use efficiency in plantswhere recovery from stress is more important than stress tolerance perse. Relative water content (RWC) is a measurement of how much (%) of theplant is water at harvest. RWC=(SWM−SDM)/(STM−SDM)×100. Fully wateredcorn plants are about 98% RWC. Typically, in a wilt screen the plantsare about 60% RWC. Plants with higher RWC at the end of a drought areconsidered to be healthier plants and more fit for post-drought recoveryand growth. Relative Growth Rate (RGR) is calculated for each shootusing the formula RGR=(SWH−SIH)/((SWH+SIH)/2)×100.

Transgenic heterozygous corn plants from multiple transgenic eventscomprising T-DNA of pMON73608, including MON87460, exhibited enhancedwater deficit stress tolerance as compared to control plants.

Example 3 Improved Field Performance of MON87460 Corn Plants under WaterDeficit

Water-limited field trials were performed using commercial grade hybridcorn in environments which received no rainfall during the target periodfor the water-deficit treatment, a span of 10 to 14 days immediatelyprior to flowering.

Two row plots, of 34 plants per row were planted at a density of 32,000plants per acre in a western Kansas location. Each transgenic plot waspaired with a non-transgenic plot of the same hybrid background. Twelvepaired-plot replicates of each of 21 independent insertion events, andits non-transgenic pair, were planted in a randomized block design.Plants were maintained in a well-watered condition using overheadirrigation until the V8 stage of development, at which time water waswithheld for a 14 day period.

On the 7th day of the water-withholding treatment the distance from thesoil surface to the tip of the youngest fully extended leaf wasdetermined for each of 3 transgene positive and transgene negativeplants in each paired plot. This measurement was repeated 5 days laterusing the same leaf as on day 7. From the day 7 and day 12 measurementsa growth rate, in cm/d, was calculated for each plant measured. Thisrate is referred to as Leaf Extension Rate (LER).

On the 8^(th) day of the treatment an estimate of chlorophyll contentwas made using the Minolta SPAD-502 (Spectrum Technologies, Plainfield,Ill.). This measurement was taken at a mid-leaf (base to tip) positionof the youngest fully expanded leaf for 6 of the 21 events. SPADreadings were collected for each of 6 transgenic positive and 6transgenic negative plants in each paired plot, for each paired plotreplicate.

Similarly, on the 8^(th) day of the treatment, photosynthetic rates weremeasured at mid-day, using the mid-leaf of the youngest fully expandedleaf for the same 6 of the 21 events. Photosynthetic rates were measuredusing the PP Systems (Amesbury, Mass.) Ciras-1 Portable PhotosynthesisSystem. Leaf photosynthesis was measured at an atmospheric [CO²] of 367mol. mol⁻¹, an ambient water vapor pressure of 2.3 kPa, and a leaf airvapor pressure deficit between 0.6 and 1.5 kPa, with photosyntheticphoton flux density between 1,200 and 1,400 mol·m⁻²·s⁻¹.

Twenty-two CspB events were evaluated in the Kansas field trial. Thewater-deficit treatment resulted in an average reduction in growth ratesto 50% of the well-watered rate. As a construct, the CspB transgenicsdemonstrated a 3.6% increase in leaf extension rates relative tonon-transgenic controls (Table 2). MON87460 and a second high performingevent demonstrated growth rate increases of 12 and 24%. The CspBpositive plants also demonstrated significant improvements inchlorophyll content and photosynthetic rates (Table 2). At a constructlevel, chlorophyll content was increased by 2.5%, with MON87460 and asecond high performing event exhibiting increases of 4.4 and 3.3%. Theimprovements to the photosynthetic rates were 3.6% at a construct level,with increases of 8.5 and 7.7% for MON87460 and a second high performingevent.

TABLE 2 Improved Growth of cspB events Under Field Water-DeficitConditions % Increase % increase LER Chlorophyll % Increase Gene-Event(field) content Photosynthesis CspB-Construct 3.6%  2.5% 3.6% CspB-ZmEvent MON87460 12% 4.4% 8.5% CspB-Zm Event 2 24% 3.3% 7.7%

Example 4 Improved Yield of MON87460 Corn Plants under Limited-WaterTreatment

The yield performance of 10 independently integrated CspB events, mostof which had previously demonstrated improved vegetative performance ineither greenhouse screens or field trials, was evaluated in an elitehybrid genetic background at 4 locations in central California where alimited-water treatment was applied. Water-limited treatment was appliedby reducing irrigation for a 14 day period during the late vegetativestage of development, immediately prior to flowering. The treatmentresulted in a net reduction of approximately 49.2 cm³ of water relativeto a well-watered regime. This was achieved by omitting two of three24.6 cm³ applications of water during the stress period. The treatmentreduced the relative growth rate during the treatment by approximately50% of well-watered rates and similarly reduced the average end ofseason grain yield by 50%. Each trial location was designed as a4-factor group unbalanced block design, and planted with 3 replicationsper location. Within each replication, the genotypes were randomized asthe 1^(st) factor, and constructs, events, and gene-positive vs.gene-negative plots were randomized as the 2^(nd), 3^(rd), and 4^(th)factors, respectively. The design placed the positive and negativeentries for each selection in adjacent 2 row plots. Final populationdensity reflected local planting practices and ranged from 65 to 76plants per 2 row plot. Plots were 21 feet long and row spacing rangedfrom 30 to 40 inches wide, reflecting local planting practices.

Grain yield data was collected from the water-limited field trials andis provided in Table 3 below. Mean yield at the water-limited Californiafields was 6.8 t/Ha, representing a 50% reduction in yield relative tothe average mean yield of crops in the Midwest. Yield averages of CspBpositive plants as a construct, were significantly greater, by 7.5%(p<0.01). A number of individual events exhibited significant yieldadvantages as well. CspB-Zm MON87460 was the best performing event anddemonstrated a yield improvement of 20.4%.

TABLE 3 Improved Yield of cspB events Under Field Water-LimitedConditions Event Yield (t/Ha) % improvement CspB Non-transgenic Mean6.86 CspB-Construct Mean 7.38  7.5% CspB-Zm Event MON87460 8.26 20.4%CspB-Zm Event 2 7.61 10.9%

The MON87460 event also demonstrated significant improvements in leafgrowth, chlorophyll content and photosynthetic rates, providing evidencethat these improvements in vegetative productivity translate intoimprovements in reproductive performance and grain yield, andidentifying MON87460 as the top performer among the multiple independenttransgenic events tested in greenhouse or field studies.

Example 5 Molecular Analysis

MON87460 was characterized by detailed molecular analyses, includingscreens for insert number (number of integration sites within the corngenome), copy number (the number of copies of the T-DNA within onelocus), the integrity of the inserted cassettes and the absence ofbackbone sequence.

Southern Blot Analyses

Approximately 2-3 g leaf tissue was dried in a lyophilizer for ˜48 hoursand ground by adding small metal beads and shaking in a paint shaker.Each sample was mixed with 6 ml extraction buffer (0.1M Tris pH 8, 0.05M EDTA, 0.5M NaCl, 1% SDS with 0.071% BME added fresh), placed in a 65°C. water bath for 45 minutes, and mixed occasionally. Potassium acetate,5M (2 ml) was added, the tubes were then inverted two times andtransferred to an ice bath for 20 minutes. Cold chloroform (3 ml) wasadded and mixed gently by inversion for 10 minutes. Samples werecentrifuged at 3500 rpm for 15 minutes. The supernatant was transferredto new tubes and combined with 4 ml cold isopropanol. Samples werecentrifuged at 3500 rpm for 15 minutes and the supernatant discarded.The pellet was resuspended in 2 ml T₅₀E₁₀ buffer with 0.1 mg/ml RNAseand incubated at 65° C. for 20 minutes. To precipitate the DNA, 3 mlisopropanol/4.4M ammonium acetate (7:1) were added to each tube andinverted to mix. Samples were centrifuged at 3500 rpm for 15 minutes andthe supernatant was discarded. The pellets were rinsed with 0.5-1.0 ml80% EtOH then transferred to microcentrifuge tubes. After a brief spinin a microcentrifuge the supernatant was discarded and the pellets wereallowed to air-dry. Pellets were resuspended in ˜200 μl TE buffer.

Approximately 10 μg of genomic DNA was digested using 100 units ofvarious restriction enzymes in a total volume of 500 μl Digests wereincubated at 37° C. overnight and EtOH precipitated. The digested DNAwas then pelleted and re-dissolved in 20 μl TE buffer. DNA probetemplates were prepared by PCR amplification of plasmid pMON73608.Approximately 25 ng of each probe was labeled with ˜100 μCi of ³²P-dCTP(Amersham catalog #AA0075) using random priming (Radprime® DNA labelingSystem, Invitrogen). Radiolabeled probes were purified using a SephadexG-50 column (Roche). Samples were loaded onto 0.8% TAE gels and run14-18 hours at 30-35V. After electrophoresis, the gels were stained in1.5 μg/ml ethidium bromide for 10-15 minutes and then photographed. Thegels were then placed in depurination solution (0.125 N HCl) for 10-15minutes followed by a denaturing solution (0.5M NaOH, 1.5 M NaCl) for30-40 minutes and then a neutralizing solution (0.5M Tris-HCl pH 7.0,1.5 M NaCl) for 30-40 minutes. The gels were then transferred to a20×SSC solution for 5-15 minutes. Capillary transfer of DNA (Southern,1975) onto Hybond-N nylon membrane (Amersham) was facilitated overnightusing a Turboblotter™ (Schleicher & Schuell) with 20×SSC transferbuffer. DNA was covalently cross-linked to the membrane with a UVStratalinker® 1800 (Stratagene) using the auto-crosslink setting andstored at 4° C. until required. Membranes were incubated for 1-4 hoursat 60-65° C. in prehybridization buffer (250 mM Na₂HPO₄.7H20 pH 7.2, 7%SDS, and 0.1 mg/ml tRNA). The ³²P-labeled probe was added to freshprehybridization buffer and hybridized overnight at 60-65° C. Membraneswere washed 3 times in an aqueous solution of 0.1% SDS and 0.1×SSC for15-20 minutes.

Probes included the intact cspB and nptII coding regions and theirrespective promoters, introns, and polyadenylation sequences and theplasmid backbone. No additional elements from the originaltransformation vector, linked or unlinked to the intact cassettes, wereidentified in the genome of these corn events. No backbone sequence wasdetected.

The data show that corn event MON87460 contains a single T-DNA insertionwith one copy of the cspB and nptII cassettes.

Results from reactions using rice actin promoter and intron sequenceprobes indicated that the full rice actin promoter sequence present inpMON73608 is not present in MON87460. The rice actin intron element wasconfirmed to be intact in MON87460.

Northern Blot Analyses

RNA from corn event MON87460 and wild type leaf tissue from greenhousegrown plants was isolated from one gram tissue samples using a ToTALLYRNA™ Kit (Ambion catalog #1910). Samples containing 5, 10, 25 and 50 μgMON87460 and wild type RNA were prepared and run on a 1.0% agarose gelat 120V for approximately 2 hours. Following electrophoresis, the gelswere then rinsed in deionized H₂0 blotted to nylon membranes. The gelswere allowed to transfer overnight. The blots were covalentlycross-linked and placed at 4° C. for short-term storage. Prior toprehybridization, the blots were pre-rinsed in 10×SSC for 2 minutes. Thebots were then placed in individual hybridization bottles with 20 mlSigma Hyb Buffer (catalog #117033) and prehybridized at 65° C. for 1hour.

Approximately 25 ng of cspB and nptII probe templates were labeled with˜50 μCi of ³²P-dCTP using random priming (Radprime® DNA labeling System,Invitrogen). Denatured cspB and nptII radiolabeled probes were thenadded to separate tubes containing 5 ml preheated hybridization buffer.The buffer containing each probe was then mixed and added to theappropriate hybridization bottle and hybridized overnight. Following anovernight hybridization the blots were removed from the bottle andplaced in low stringency wash buffer (2×SSC, 0.1% SDS) in a glass trayand placed on a shaker for 10 minutes at room temperature. The blotswere placed on blotting paper and then in fresh hybridization bottleswith 25 ml of low stringency pre-warmed wash buffer (65° C.). The blotswere washed at 65° C., two times in low stringency wash buffer for 15minutes and once at 65° C., in high stringency wash buffer (0.5×SSC,0.1% SDS) for 15 minutes.

Northern blot analysis confirmed that the expected size transcripts forboth cspB (−600 nt) and nptII (˜1100 nt) are generated in MON87460.

Sequencing T-DNA Insert and Flanking Corn Genomic DNA in Lambda Clone

High quality genomic DNA from corn event MON87460 was isolated using anSDS chloroform extraction method. MON87460 genomic DNA was digested withMfeI and purified using the QIAEX® II Gel Extraction Kit (Qiagen), toensure the purification of fragments greater than 10 kb. This digestedand purified genomic DNA was used for ligation into the Lambda DASH®II/EcoRI Vector Kit (Stratagene). Approximately 2.5×10⁵ colonieswerescreened using ³²P-labeled Ract intron and cspB probes. Purified DNAfrom a pure bacteriophage lambda clone was used as template insequencing reactions to confirm the T-DNA nucleotide sequence of theMON87460 insert and corn genomic DNA flanking the 5′ and 3′ ends of theMON87460 insert.

DNA sequence analysis confirms that the in planta T-DNA in MON87460 isidentical to the corresponding sequence in pMON73608. This sequenceanalysis also characterized the extent of the truncation of the 5′ endof the rice actin promoter which had been observed in Southern analysis.The sequence analysis revealed that the Agrobacterium RB and most of theP-ract promoter are not present in the MON87460 event. The P-ractpromoter in MON87460 consists of only 108 bp of the 3′ end of the fullrice actin promoter region (˜850 nt) in pMON73608. This result alsoconfirms that the in planta sequence for cspB and nptII in corn eventMON87460 match the exact coding regions within the transformation vectorpMON73608. This clone also confirmed 1060 bp of 5′ flanking sequence,3309 bp of T-DNA insert and 1260 bp of 3′ flanking sequence for theMON87460 insert.

Wildtype Allele Analysis

PCR was performed on genomic DNA from the nontransgenic corn line usedin transformation using primers that hybridize to the 5′ and 3′ flankingregions of the MON87460 insert. Multiple primer combinations wereperformed with each combination consisting of a primer that hybridizesto the 5′ and 3′ flanking region, respectively. The PCR analysis wasperformed using ˜50 ng of genomic DNA template in a 50 μl reactionvolume. Resulting amplicons were then sequenced. Analysis of the wildtype allele showed that a 22 bp deletion of corn genomic DNA (SEQ IDNO:23) occurred upon integration of the MON87460 T-DNA into the cornchromosome.

Example 6 Detection of MON87460 Event Polynucleotides

The detection of MON87460 event in progeny resulting from breeding witha MON87460 line may be accomplished by extraction of genomic DNA fromcorn plant tissues and analysis for MON87460 specific polynucleotides.Of particular interest for identification of MON87460 polynucleotides isthe use of PCR to amplify genomic DNA comprising transgene/genomicjunction sequences.

An amplicon diagnostic for MON87460 comprises at least one junctionsequence, SEQ ID NO: 1 or SEQ ID NO: 2 (FIG. 2). SEQ ID NO: 1corresponds to the junction of the arbitrarily designated 5′ flankingsequence (positions 1051 through 1060 of SEQ ID NO: 5) and the 5′ regionof the truncated rice actin promoter (positions 1-10 of SEQ ID NO:7) inthe cspB expression construct. SEQ ID NO: 2 corresponds to the junctionof the integrated left border from pMON73608 (positions 3300 through3309 of SEQ ID NO: 7) and the arbitrarily designated 3′ flankingsequence (positions 1 through 10 of SEQ ID NO: 6).

Event primer pairs that will produce a diagnostic amplicon for MON87460include primer pairs based upon the flanking sequences and the insertedDNA from pMON73608. To generate a diagnostic amplicon comprising atleast 11 nucleotides of SEQ ID NO: 1, a forward primer based upon SEQ IDNO: 5 and a reverse primer based upon the inserted transgene sequence,SEQ ID NO: 7 are prepared. Similarly, to generate a diagnostic ampliconcomprising at least 11 nucleotides of SEQ ID NO: 2, a forward primerbased upon inserted transgene sequence, SEQ ID NO: 7, and a reverseprimer based upon the 3′ flanking sequence, SEQ ID NO: 6 are prepared.It is readily apparent to one skilled in the art the primer pairs mayalso be designed to produce an amplicon comprising polynucleotidescomplementary to at least 11 nucleotides of SEQ ID NO:1 or SEQ ID NO:2,in which case the forward and reverse sequences are based upon sequencescomplementary to those in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

Primers are designed which produce amplicons having between 50 and 1000bases. Amplification conditions are as illustrated in Table 4 and Table5 below and include a positive tissue control from event MON87460, anegative control from a corn plant that is not event MON87460, and anegative control that contains no corn genomic DNA. A primer pair thatwill amplify an endogenous corn DNA molecule, such as from the ADH gene,may be used as an internal control for the DNA amplification conditions.

Corn plant DNA for use in DNA amplification reactions may be isolatedfrom any suitable corn plant tissue, and is preferably isolated fromnewly formed leaf tissue from plants <1 month old for reactions asdescribed herein. Leaf tissue is harvested using a standard 7 mm holepunch, to collect tissue equivalent to an approximately 1 centimeterwide and 1 inch long leaf tear. Tissue samples are lyophilized and driedtissue is ground by adding 4-6 3 mm zirconia-silica beads to each tissuesample in a polypropylene tube and shaking in a paint shaker.Homogenized tissue samples are mixed in a 96-well plate with 395 ul ofpre-warmed SDS extraction buffer (0.1M Tris pH 8, 10 mM EDTA, 1.0 MNaCl, 1% SDS), vortexed briefly and incubated at 65° C. for 45 minutes.135 ul of cold potassium acetate (5M) is added. Samples are mixed byvortexing and the plate is spun at 3300 rpm for 20 minutes. 100 ul ofsupernatant is transferred to a fresh 96-well plate containing 100 ulisopropanol and samples are vortexed to mix. The plate is spun at 3300rpm for 20 minutes and the supernatant discarded. Plates are drainedupside down for 1 minute. 300 ul of cold 70% ethanol is added and theplate is vortexed briefly and placed at 4° C. for 30 minutes. The plateis spun at 3300 rpm for 20 minutes and supernatant is discarded. Theplate is drained upside down and the ethanol precipitation repeated.After a final spin (3300 rpm 20 minutes), the plate is drained for oneminute and placed on its side in a 65° C. oven for about 15-30 minutesto dry the pellet. The DNA is resuspended in 100 ul pH 8.0 TE buffer(Sigma) containing RNase (10 ug/ml. DNA is stored at 4° C. overnight.DNA yield is about 1 ug (10 ng/ul).

The assay for the MON87460 amplicon can be performed using an AppliedBiosystems GeneAmp PCR System 9700, Stratagene Robocycler, MJ Engine,Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermocycler or anyother amplification system that can be used to produce an amplicondiagnostic of MON87460.

TABLE 4 Corn MON87460 Event Specific PCR Step Reagent Volume Comments 118 megohm water adjust for final volume of 10 ul 2 2X Universal MasterMix 5.0 ul 1X final concentration of (Contains dNTPs, enzyme and buffer)dNTPs, enzyme and buffer 3 Primer-1 and Primer-2 Mix (resuspended in 180.5 ul 1.0 uM final concentration megohm water to a concentration of 20uM for each primer) Example: In a microcentrifuge tube, the followingshould be added to achieve 500 ul at a final concentration of 20uM: 100ul of Primer 1 at a concentration of 100 uM 100 ul of Primer 2 at aconcentration of 100 uM 300 ul of 18 megohm water 4 Extracted DNAtemplate (5-10 ng each): 3.0 ul Leaf samples to be analyzed Negativecontrol (non-transgenic DNA) Negative water control (no templatecontrol) Positive control (MON87460 DNA)

TABLE 5 Endpoint TaqMan thermocycler conditions Cycle No. Settings 1 50°C.  2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64° C.  1 minute(−1° C./cycle) 30 95° C. 15 seconds 54° C.  1 minute 1 10° C. Forever

Amplicons produced using the designed primer pairs are shown to containMON87460 polynucleotides by hybridization to probes specific forMON87460 junction sequences SEQ ID NO:1 or SEQ ID NO:2, or by isolationand DNA sequence analysis.

Example 7 Endpoint TaqMan Event-Specific Assay

A MON87460 event-specific endpoint TaqMan PCR reaction is describedherein. With Endpoint Taqman, the signal corresponding to a particularamplification is quantified using a fluorescent detection system afterthe reaction cycling is complete. The use of three site-specifichybridizations (two PCR primers and a fluorescently labeled probe) forsignal generation provides a highly specific assay. The probe anneals tospecific nucleotides between the forward and reverse primers. Whennucleotide extension reaches the hybridized probe, taq polymerasedegrades the probe releasing the fluor from the quencher so that asignal is emitted. The signal is read after the reactions are complete.

Polynucleotide primers used in the endpoint assay are primers SQ10443(SEQ ID NO: 8), SQ10445 (SEQ ID NO: 9) and the probe used to detect theMON87460 amplicon is 6FAM™ labeled MGBNFQ (minor groove binding, nonfluorescent quencher) probe PB3814 (SEQ ID NO: 10). An internal corn DNAprimer may also be used to confirm integrity of the template DNA. Forexample, amplification of alcohol dehydrogenase (ADH), a single-copyendogenous gene within the corn genome, may be accomplished usingprimers SQ5263 (SEQ ID NO:11) and SQ5264 (SEQ ID NO:12) and detectedwith VIC™ (reporter fluorochrome) and TAMRA™ (quencher fluorochrome)probe PB2033 (SEQ ID NO:13). 6FAM™, VIC™ and TAMRA™ are fluorescent dyeproducts of Applied Biosystems (Foster City, Calif.) attached to the DNAprobes. In these analyses, Taq DNA polymerase cleaves probes thatspecifically hybridize to the amplified DNA and releases thefluorophore. The separation of fluorophore and quencher allowsfluorescence to occur which is diagnostic under these conditions for thepresence of MON87460 polynucleotides.

SQ10443 (SEQ ID NO: 8) and SQ10445 (SEQ ID NO: 9) when used as describedin Table 2 below produce a 68 nt DNA amplicon (SEQ ID NO:20) that isdiagnostic for event MON87460 DNA and detected by hybridization to apolynucleotide probe, such as PB3814. This assay has been optimized foruse in 96-well or 384-well format using an Applied Biosystems GeneAmpPCR System 9700 or MJ Research DNA Engine PTC-225. Other methods andapparatus may be known to those skilled in the art and used to produceamplicons that identify the event MON87460 DNA. Adjustments to cyclingparameters may be needed to ensure that ramp speeds are equivalent. Cornleaf tissue samples are used in the below analysis, and should bethoroughly ground to produce a homogenous sample. Corn leaf DNA isisolated as described in Example 6. The concentration of the leaf DNA tobe tested is preferably within the range of 5-10 ng per PCR reaction.Control DNA should be extracted using the same method as for extractionof the samples to be analyzed. Controls for this analysis should includea positive control from corn known to contain event MON87460 DNA, anegative control from non-transgenic corn and a negative control thatcontains no template DNA.

For PCR reactions using an Applied Biosystems GeneAmp PCR System 9700 orMJ Research DNA Engine PTC-225 thermal cycler, cycling parameters asdescribed in Table 3 below are used. When running the PCR in thePerkin-Elmer 9700, the thermocycler is run with the ramp speed set atmaximum.

TABLE 6 Corn MON87460 Event Specific Endpoint TaqMan PCR Step ReagentVolume Comments 1 18 megohm water adjust for final volume of 10 ul 2 2XUniversal Master Mix 5.0 ul 1X final concentration of (Contains dNTPs,enzyme and buffer) dNTPs, enzyme and buffer 3 Primer-1 and Primer-2 Mix(resuspended in 18 0.5 ul 1.0 uM final concentration megohm water to aconcentration of 20 uM for each primer) Example: In a microcentrifugetube, the following should be added to achieve 500 ul at a finalconcentration of 20uM: 100 ul of Primer SQ10443 at a concentration of100 uM 100 ul of Primer SQ10445 at a concentration of 100 uM 300 ul of18 megohm water 4 Event 6-FAM ™ MGBNFQ Probe PB3814 0.2 ul 0.2 uM finalconcentration (resuspended in 18 megohm water to a concentration of 10uM) 5 Internal Control Primer-1 (SQ5263) and Internal 0.5 μl 1.0 μMfinal concentration Control Primer-2 (SQ5264). Mix (resuspended in 18megohm water to a concentration of 20 μM for each primer) 6 InternalControl VIC ™ Probe (PB2033; SEQ 0.2 μl 0.2 μM final concentration IDNO: 13) resuspended in 18 megohm water to a concentration of 10 μM 7Extracted DNA template (5-10 ng each): 3.0 ul Leaf samples to beanalyzed Negative control (non-transgenic DNA) Negative water control(no template control) Positive control (MON87460 DNA)

TABLE 7 Endpoint TaqMan thermocycler conditions Cycle No. Settings 1 50°C.  2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64° C.  1 minute(−1° C./cycle) 30 95° C. 15 seconds 54° C.  1 minute 1 10° C. Forever

Example 8 Endpoint TaqMan PCR Zygosity Assay

A specific assay is described to detect the presence and zygosity(homozygous or hemiozygous) of MON87460 transgenic event in genomic DNAextracted from corn leaf tissue as described in Example 6. Determiningzygosity for event MON87460 in a sample was done using an event-specificzygosity endpoint TaqMan PCR for which examples of conditions aredescribed in Table 8 and Table 9. The DNA primers and probes used in thezygosity assay are primers SQ21105 (SEQ ID NO: 14) and SQ21106 (SEQ IDNO: 15), and 6FAM™ labeled MGB (minor groove binding) probe PB3771 (SEQID NO:16) for detection of MON87460 junction polynucleotides, andprimers SQ21195 (SEQ ID NO:17 and SQ21196 (SEQ ID NO:18), and VIC™labeled MGB probe PB 10223 (SEQ ID NO:19) for detection of wild-typecorn DNA at the insertion site.

SQ21105 (SEQ ID NO: 14) and SQ21106 (SEQ ID NO: 15) when used in thesereaction methods with PB3771 (SEQ ID NO:16) produce a 134 nt labeled DNAamplicon (SEQ ID NO:21) that is diagnostic for event MON87460 DNA.SQ21195 (SEQ ID NO:17 and SQ21196 (SEQ ID NO:18), when used in thesereaction methods with PB2512 (SEQ ID NO: 12) produce a 145 nt DNAamplicon (SEQ ID NO:22) that is diagnostic for the wild type allele. Theprobe for this reaction is specific to the 22 bp deletion of genomic DNA(SEQ ID NO:23) that occurred at the MON87460 insertion site.Heterozygosity is determined by the presence of both amplicons asdemonstrated by the liberation of fluorescent signal from both probesPB3771 and PB 10223. Homozygous corn plant genetic material isidentified by liberation of only the 6FAM™ signal from PB3771. Controlsfor this analysis should include a positive control from corn plantsamples homozygous and hemizygous for event MON87460 DNA, a negativecontrol from non-transgenic corn, and a negative control that containsno template DNA.

This assay has been optimized for use in 96-well or 384-well formatusing an Applied Biosystems GeneAmp PCR System 9700 or MJ Research DNAEngine PTC-225. When running the PCR in the MJ Engine, the thermocyclershould be run in the calculated mode. When running the PCR in thePerkin-Elmer 9700, the thermocycler is run with the ramp speed set atmaximum.

TABLE 8 Corn MON87460 Event-Specific Zygosity Endpoint TaqMan PCR StepReagent Volume Comments 1 18 megohm water adjust for final volume of 10ul 2 2X Universal Master Mix 5.0 ul 1X final concentration of (ContainsdNTPs, enzyme and buffer) dNTPs, enzyme and buffer 3 Primer-1 andPrimer-2 Mix (resuspended in 18 0.5 ul 1.0 uM final concentration megohmwater to a concentration of 20 uM for each primer) Example: In amicrocentrifuge tube, the following should be added to achieve 500 ul ata final concentration of 20uM: 100 ul of Primer SQ21105 at aconcentration of 100 uM 100 ul of Primer SQ21106 at a concentration of100 uM 300 ul of 18 megohm water 4 Event 6-FAM ™ MGB Probe PB3771 0.2 ul0.2 uM final concentration (resuspended in 18 megohm water to aconcentration of 10 uM) 5 Wild-type Primer-1 (SQ21195) and Wild-type 0.5μl 1.0 μM final concentration Primer-2 (SQ21196) Mix (resuspended in 18megohm water to a concentration of 20 μM for each primer) 6 Wild-typeVIC ™ MGB Probe (PB10223) 0.2 μl 0.2 μM final concentration resuspendedin 18 megohm water to a concentration of 10 μM 7 Extracted DNA template(5-10 ng each): 3.0 ul Leaf samples to be analyzed Negative control(non-transgenic DNA) Negative water control (no template control)Positive control (Homozygous MON87460 DNA) Positive control (HemizygousMON87460 DNA)

TABLE 9 Zygosity Endpoint TaqMan thermocycler conditions Cycle No.Settings 1 50° C.  2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds64° C.  1 minute (−1° C./cycle) 30 95° C. 15 seconds 54° C.  1 minute 110° C. Forever

Example 9 MON87460 Yield Performance

Additional field trials were conducted with CspB expressing event,MON87460, to further investigate the ability of this event to providetolerance to water-deficits during the late vegetative and reproductivedevelopmental stages. These are very important stages from anagricultural perspective due to the sensitivity of the crop at thesegrowth stages and the frequency with which a drought occurs during thesedevelopmental stages in the growing regions targeted.

Yield performance of MON87460 was evaluated in three elite hybridgenetic backgrounds at 5 replicated locations across central Californiaand western Kansas where two distinct limiting-water treatments wereapplied. The late vegetative treatment was applied to the trials byreducing irrigation for a 14 day period during the late vegetative stageof development, immediately prior to flowering. The treatment reducedthe relative growth rate during the treatment by approximately 50% ofwell-watered rates and similarly reduced the average end of season grainyield by 50%. A grain fill treatment was achieved by initiating thewater-limiting conditions at a later stage, relative to the vegetativetreatment, depleting the soil moisture profile on or around floweringand achieving maximal stress during the grain fill period. Thistreatment resulted in an approximate 25% reduction in plant heights anda 30-40% reduction in grain yield as a result of the stress imposition.Three hybrids expressing the CspB event were evaluated using 20replications of data across 5 locations for each stress treatmentwindow.

Each trial location was designed as a 3 factor group unbalanced blockdesign, and planted with 4 replications per location. Within eachreplication, the genotypes were randomized as the 1^(st) factor, andevents, and gene-positive vs. gene-negative plots were randomized as the2^(nd), and 3^(rd) factors, respectively. The design placed the positiveand negative entries for each selection in adjacent 2 row plots. Finalpopulation density reflected local planting practices and ranged from 65to 76 plants per 2 row plot. Plots were 21 feet long and row spacingranged from 30 to 40 inches wide, reflecting local planting practices.

Analysis of the yield data was performed using Version 9.1.3 of SAS/STATsoftware (SAS Institute Inc., 2003). Analysis of variance calculationswere performed using the MIXED and GLIMMIX procedures. Outliers wereidentified individually at each location by calculating the deletedStudentized residuals with respect to the corresponding linear model fora single location, comparing those residuals to zero using t-tests at anexperiment wise Type I error rate of 5% using Bonferroni-adjustedp-values, and removing the identified outliers. After two passes throughthe data, all remaining observations were included in the analyses.Yield was determined for each plot, and analyzed using a mixed modelwith fixed effects for constructs and events nested within constructsand random effects for locations, reps within locations, and theinteraction of locations with constructs. These analyses were performedseparately for each hybrid. Comparisons of event and construct averagesto negative paired entries were made with t-tests applied toleast-squares means. Yield stability was examined by comparing simplelinear regression estimates derived from positive and negative events.In both cases, the regression model included the average yield of theevent at a location as the response and the average yield of acommercial check pedigree at the same location as the predictor.Positive and negative entries were then compared by using theirpredicted yields from the regression model at various benchmark yieldsof the commercial check.

MON87460 corn plants exhibited improvements in end of season grain yieldacross the different hybrid entries and under both water stress regimeswhen compared to a conventional wild-type control of the same geneticbackground (Table 10). Yield benefits in these experiments ranged from11% to as much at 21% across yield values that averaged 6.4 to 8.5 t/Ha.The transgenic CspB event consistently out-yielded the non-transgeniccontrols by at least 0.5 t/Ha across 12 out of 15 reproductive stresstreatments and 13 out of 15 vegetative stress treatments.

TABLE 10 MON87460 Yield Results From Managed Irrigation Water-deficitConditions Mean Yld Mean Yld t/Ha % dif- Stress class Entries Pos (t/Ha)Check (t/Ha) difference ference Vegetative Hybrid 1 10.1 8.5 1.6 19(positive) Reproductive Hybrid 1 9.0 7.7 1.3 16 (positive) All StressHybrid 1 9.1 7.9 1.1 14 (positive) Vegetative Hybrid 2 7.7 6.5 1.2 18(positive) Reproductive Hybrid 2 8.1 6.8 1.3 19 (positive) All StressHybrid 2 7.7 6.4 1.3 21 (positive) Vegetative Hybrid 3 8.3 7.2 1.1 16(positive) Reproductive Hybrid 3 8.9 8.0 0.9 11 (positive) All StressHybrid 3 8.8 7.9 0.9 12 (positive)

A multi-year analysis was also conducted with MON87460 to assess thestability of the yield advantages across locations under water-limitingconditions. Locations that had experienced some level of water stress,where yield reductions ranged from 20 to 80%, were compiled andanalyzed. Yield advantages were evident across multiple years of testingand under a wide range of environments with varying degrees ofwater-deficit stress.

Across four years of testing, MON87460 has demonstrated an average yieldbenefit of 10.5% across three hybrid test-crosses under managed stressenvironmental testing. The average yield advantage each year was 0.89,0.48, 0.49 and 0.79 t/Ha, representing percentage increases of 13.4,6.7, 10.5 and 11.3%, respectively.

Dryland market evaluations of MON87460 hybrid entries were conducted inthe states of South Dakota, Nebraska, and Kansas. Locations wereselected on the basis of historical weather patterns and average countyyields of 4.5 to 7.7 t/Ha. Each trial location was designed as asplit-plot unbalanced block design and planted with a single replicationper location. Plots were 100 feet long and four rows wide and finalpopulation densities reflected local planting practices undernon-irrigated conditions of approximately 200 plants per 100 foot row.Row spacing ranged from 30 to 40 inches wide, reflecting local plantingpractices. Weather stations were installed at each location and thetrials were monitored for signs of water-deficit stress throughout theseason. No supplemental water was provided. Environmental data wascollected and seasonal weather patterns, including rainfallaccumulation, were utilized to classify the water-deficit stress duringthe season for each dryland location. 12 of the locations planted acrossthese three states were categorized as having experienced water stressduring the late vegetative through reproductive developmental stages andwere utilized for analysis.

Yield benefits were observed in the same three hybrid backgrounds thatwere evaluated under controlled water-deficit conditions described inTable 10. When compared to the non-transgenic control, the MON87460event provides yield benefits of up to 0.75 t/Ha, or 15%. These drylandgrowing conditions created a lower yielding environment (average yieldof the controls were 4.9 t/Ha) than the controlled water-deficitlocations where the overall yields of the controls ranged from 6.4 to8.5 t/Ha.

Thus, significant yield improvements are obtained with MON87460 undercontrolled drought environments as well as under water stressed westerndryland conditions. MON87460 provides water stress tolerance by usingwater more efficiently than negative controls by delivering improvedgrowth rates and grain yields under water stress conditions while usingequivalent or less water.

Example 10 Plant Breeding to Produce Herbicide Tolerant MON87460 Plants

MON87460 event plants are crossed with a herbicide tolerant corn plantexpressing a glyphosate resistant 5-enol-pyruvylshikimate-3-phosphatesynthase (EPSPS) gene to generate improved plants having both waterdeficit tolerance and herbicide tolerance. Of particular interest is across of a MON87460 event corn plant to a herbicide tolerant corn eventplant designated as event PV-ZMGT32(nk603) and described in U.S. Pat.No. 6,825,400.

Crossing is conducted with two homozygous inbred lines, one of M0N87460and one of PV-ZMGT32(nk603) to produce hybrid seed for commercialplanting of a corn crop having water deficit and herbicide tolerance.

Alternatively, a single inbred line comprising both MON87460 andPV-ZMGT32(nk603) is generated using a recurrent parent backcrossingbreeding method to produce a fixed line homozygous for both traits. Theinbred line developed in this manner exhibits water deficit toleranceand herbicide tolerance traits. The inbred line is crossed with a secondinbred line, which may be an elite wild type line or a transgenic eventline demonstrating one or more improved traits, to produce hybrid seedfor planting to produce an improved corn crop.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations can be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1.-28. (canceled)
 29. A polynucleotide comprising SEQ ID NO:25, or a complement thereof. 30.-33. (canceled)
 34. A probe comprising the polynucleotide of SEQ ID NO:25, or a complement thereof, wherein a detectable label or a reporter molecule is attached to said polynucleotide.
 35. A corn seed comprising SEQ ID NO:25.
 36. A processed food or feed commodity comprising the polynucleotide of SEQ ID NO:25.
 37. The processed food or feed commodity of claim 36, wherein said food or said feed commodity comprises corn meal, corn flour, corn gluten, corn oil, or corn starch.
 38. A method of producing water deficit stress tolerant corn plants comprising the step of crossing a first parental homozygous corn plant of event MON87460 having the polynucleotide of SEQ ID NO:25 with a second parental homozygous corn plant that lacks the water deficit stress tolerance trait, thereby producing water deficit stress tolerant hybrid progeny plants.
 39. The method of claim 38, wherein anyone of SEQ ID NO:1-4, 5-7, or 25 are used to identify the water deficit stress tolerant hybrid progeny plants.
 40. The method of claim 38, wherein said second parental homozygous corn plant is homozygous for an exogenous gene.
 41. The method of claim 40, wherein the exogenous gene expresses a glyphosate resistant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein.
 42. A corn plant comprising SEQ ID NO:25. 